The present invention relates to cloning and recombinant expression of proteins. In particular, it relates to expression of vanadium haloperoxidase polypeptides.
Vanadium haloperoxidase enzymes are useful in industrial catalysis in a variety of contexts (Sheffield, et al., Biotechnology Techniques, 8:579-582 (1994)). For instance, they catalyze a variety of halogenation, oxidation and epoxidation reactions (Itoh, et al., Eur. J. Biochem., 172:477 (1988); Itoh, et. al. Biochimica et Biophysica Acta., 1994 (1993); Itoh, et al., Appl. Microbiol. and Biotechnol., 43:394-401 (1995)). Although a halide ion is a required cofactor for enzyme activity, products may not be halogenated. Numerous uses in synthetic organic chemistry include reactions with diverse substrates such as aliphatic and aromatic hydrocarbons, phenols, xcex2-diketones and nitrogen- and sulfur-containing heterocycles (Itoh, et al., Eur. J. Biochem. 172:477 (1988); Neidleman et al., Biohalogenation: Principles, Basic Roles and Applications, Ellis Horwood, John Wiley and Sons, New York (1986)). Bromoperoxidases can also be used in place of synthetic organic chemistry reactions to make activated intermediates or products such as pesticides. In addition, these enzymes have an advantage over chemical synthesis in producing stereospecific products (Itoh, et al., Eur. J. Biochem., 172:477 (1988)). Moreover, haloperoxidases have unusual stability (both temporal and thermal) and are active in solvents including methanol, ethanol and acetone.
Recent medical applications of bromoperoxidase have been described. Lovqvist, et al., Nuclear Medicine and Biology, 22:125-131 (1995) described the enzymatic bromination of a monoclonal antibody with BR-radionuclide for imaging of antibody localization by PET scanning. There is current interest in enzymatic production of antibiotics including fosfomycin and pyrrolnitrin (Itoh, et. al. Biochimica et Biophysica Acta. 1994 (1993); Itoh, et al., Appl. Microbiol. and Biotechnol. 43:394-401 (1995)) and 7-chlorotetracycline (van Pxc3xa9e, K. H., J. Bacteriol., 170:5890-5894 (1988)) via haloperoxidase-catalyzed reactions in bacteria.
Known haloperoxidases include bromoperoxidases from brown and red algae including Fucus and Ascophyllum (Butler, et al., Chem. Rev., 93:1937-1994 (1993)), iodoperoxidase from green algae (Mehrtens, G., Polar Biol. 14:351-354 (1994)), and chloroperoxidase from the fungus Curvularia inaequalis (Van Schijndel, et al., Eur. J. Biochem., 225:151-157 (1994)). A vanadate requirement for algal haloperoxidase was first described by Vilter (Vilter, H., Biological Systems, 31, Vanadium and its role in life, Sigel, et al. (Eds.), Marcel Dekker, New York, N.Y., pp. 325-362 (1995)).
The specific bromoperoxidase activity of the native Fucus enzyme is several fold higher (Butler, et al.) than the other algal enzymes for which at least partial sequences have been reported, Ascophyllum (Vilter 1995) and Corallina (Shimonishi, et al. FEBS Letters, 428, 105-110 (1998)), and higher specific activity than the Curvularia fungal chloroperoxidase (van Schijndel et al. BBA 1161:249-256 (1993)).
Extracted and partially purified bromoperoxidase from the red alga Corallina officinalis is commercially available from Sigma Chemical Company. Sigma has also investigated immobilization of enzyme on agarose beads (Sheffield, et al., Phytochemistry, 38:1103-1107 (1995)) and on cellulose acetate membrane (Sheffield, et al., Biotechnology Techniques, 8:579-582 (1994)) for repetitive catalysis of bromination reactions in flow-through reactors in enzyme-driven preparative organic chemistry. Many industrial uses for stable soybean peroxidase are envisioned by A. Pokora of Enzymol International, Inc. as described by Wick (Wick, C. B., Genetic Engineering News, 16(3): 1, 18-19). Recombinant enzyme biotechnology is of current industrial interest because enzymes are safe, low-polluting alternatives to chemicals in many applications, and can be modified by protein engineering to fit the requirements of specific applications (Kelly, E. B. Genetic Engineering News, 16(5):1, 30, 32 (1996) Lovqvist, et al., Nuclear Medicine and Biology, 22:125-131 (1995)). Peroxidases can also be incorporated into moldable plastics (Service, R. F., Science 272:196-197 (1996)).
Multiple representatives of other classes of peroxidases have been produced in recombinant form. A heme peroxidase, manganese peroxidase from the fungus Phanerochaete chrysosporidium, was expressed in recombinant form and refolded for activity (Whitwam, R. E., Biochem. Biophys. Research. Communications, 216:1013-1017 (1995)). Recombinant horseradish peroxidase isozyme C (a heme peroxidase) for use in chemiluminescent labeling in molecular biology and biotechnology applications has been described (EP 0299682, WO 89/03424). Recombinant non-heme haloperoxidases have been prepared from the bacteria, Pseudomonas pyrrocinia (Wolfframm, et al., Gene 130:131-135 (1993)) and two related Streptomyces aureofaciens enzymes (van Pee, K. H., J. Bacteriol., 170:5890-5894 (1988); Pfeifer, et al., J. Gen. Microbiol. 138:1123-1131 (1992)).
The X-ray structure for the fungal chloroperoxidase from Curvularia has been reported Messerschmidt and Wever., Proc. Natl. Acad. Sci. USA. 93:392-396 (1996). The chloroperoxidase consisted of two helical bundle domains organized as an N-terminal noncatalytic bundle and a C-terminal catalytic bundle. The X-ray structure of the bromoperoxidase from the brown alga Ascophyllum (Weyand et al J. Mol. Biol. 293:595-611 (1999)) and the bromoperoxidase from the red alga Corallina (Littlechild Curr. Opin. Chem. Biol. 3:28-34 (1999)) have been published.
Despite the interest in vanadium haloperoxidases, there are relatively few reports in the literature of the cloning and recombinant expression of a vanadium haloperoxidases. Shimonshi et al. FEBS Lett. 428:105-110 (1998) described cloning of the enzyme from Corallina pilulifera. Cloning of the Curvularia gene is described by Hemrika, et al. PNAS 94,2145-2149 (1997) and 95/27046. A partial sequence of the Ascophyllum gene is described in Vilter (1995). There is a need in the art for efficient means for producing vanadium haloperoxidases using techniques such as recombinant expression. The present invention addresses these and other needs.
The present invention provides isolated nucleic acids comprising a polynucleotide sequence encoding a vanadium haloperoxidase polypeptide consisting of a catalytic helical frame that complexes a vanadium ion and catalyzes the oxidation of o-dianisidine (ODA). The polypeptides of the invention preferably comprise amino acids unique to the Fucus haloperoxidase. Examples include an Ala residue at a position corresponding to position 455 of SEQ ID NO: 2, a Cys residue at a position corresponding to position 457 of SEQ ID NO:2, or a Val residue at position 525 of SEQ ID NO: 2.
In some embodiments, the haloperoxidase polypeptide comprises an amino acid sequence having at least about 70% amino acid sequence identity to an amino acid sequence from residue 435 to residue 632 as set forth in SEQ ID NO:2. The polypeptide may have a molecular weight of about 20 kDa. The isolated nucleic acid will usually have a polynucleotide sequence at least about 70% sequence identity to a subsequence as of SEQ ID NO:1.
To facilitate recombinant expression, the polynucleotide sequence is often included in a recombinant expression cassette in which the polynucleotide sequence is operably linked to a promoter sequence. The invention also provides cells comprising the expression cassette of the invention.
In some embodiments, the polypeptides of the invention are immobilized on a solid surface. The polypeptide may further comprise a cleavable linker sequence, such as an enterokinase cleavable linker sequence. The polypeptide may also further comprise an purification tag, such as a plurality of histidine residues.
The invention also provides method for enzymatically halogenating or oxidizing a compound using the enzymes of the invention.
The invention further provides method for preparing active vanadium haloperoxidase polypeptides of the invention using preferred methods of refolding. The refolding may comprise contacting the vanadium haloperoxidase polypeptide with an ammonium sulfate solution with or without magnesium sulfate. In other embodiments, the method use magnesium sulfate, preferably at about 0xc2x0 C. to about 10xc2x0 C. Still further methods of refolding comprise contacting the vanadium haloperoxidase polypeptide with imidazole and sodium or potassium chloride, preferably at about 10xc2x0 C. to about 17xc2x0 C.
A xe2x80x9cvanadium haloperoxidase polypeptidexe2x80x9d of the invention is an isolated protein capable of catalyzing the oxidation of o-dianisidine (ODA) when complexed with a vanadium ion. Vanadium haloperoxidases of the invention can also be identified by the presence of a catalytic frame helical motif (sometimes referred to herein as a xe2x80x9ccatalytic bundlexe2x80x9d) exemplified by residues 435 to residue 631 in SEQ ID NO:2. The catalytic frame motif of a haloperoxidase comprises the helices immediately part of or adjacent to the three conserved vanadium-binding regions identified below. The fourth helix of the catalytic frame can be either upstream of the equivalent of xcex11 in the Fucus sequence (as in the fungal haloperoxidases wherein the four helical frame is composed of helices K, L, N, and O and L, N and O are associated with vanadate binding site) or equivalent to xcex14 in the Fucus sequence. Polypeptides of the invention typically have a sequence at least about 70% identical (as determined below), usually at least about 80% identical to the sequence from residue 435 to residue 631 in SEQ ID NO:2. One of skill will recognize that the sequence of the polypeptide can be altered without substantially altering activity of the polypeptide (e.g., by conservative substitutions). In addition, as explained below, less conservative modifications (e.g., substitutions, additions, and deletions) can be made to facilitate proper refolding, purification, and the like, as desired.
Full length vanadium haloperoxidase polypeptides of the invention typically have a mass of about 73.4 kDa, and have a sequence as shown in SEQ ID NO:2. One of skill will recognize that shorter vanadium haloperoxidase polypeptides can also be used. For instance, the polypeptides can consist essentially of the C terminal region described above. The polypeptides may thus comprise from about 90 amino acids to about 300 amino acids, or from about 120 to about 250 amino acids. Exemplary polypeptide having a mass of about 20 kDa or less are described in detail below.
A xe2x80x9cpolynucleotide sequence encoding a vanadium haloperoxidase polypeptidexe2x80x9d of the invention is a polynucleotide which encodes a vanadium peroxidase polypeptide as described above. Thus, the nucleic acids of the invention can be altered by substitutions, deletions, and additions, as desired. Polynucleotide sequences of the invention will typically be at least about 60%, usually at least about 70%, more usually at least about 80%, and often at least about 90% or 95% identical to a subsequence of SEQ ID NO:1 which encodes 435 to residue 631 in SEQ ID NO:2. Polynucleotides of the invention can also be identified by their ability to hybridize under defined conditions to such a nucleic acid. Means for determining this are described in detail below.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual organism or cell is a polynucleotide which is introduced into the organism or cell using genetic engineering techniques.
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
A xe2x80x9cpromoterxe2x80x9d is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A xe2x80x9cconstitutivexe2x80x9d promoter is a promoter that is active under most environmental and developmental conditions. An xe2x80x9cinduciblexe2x80x9d promoter is a promoter that is active under environmental or developmental regulation. The term xe2x80x9coperably linkedxe2x80x9d refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. In addition, the term specifically includes those sequences substantially identical (determined as described below) with polynucleotide sequences disclosed here.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid. substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably at least about 70% or 80%, most preferably 90 or 95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A xe2x80x9ccomparison windowxe2x80x9d, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altshul, et al., J. Mol. Biol., 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. One of skill will recognize, however, that conservative amino acid substitutions may still affect the properties of the protein in terms of polarity, hydrophobicity, enzymatic activity, and the like. Similarly, less conservative amino acid substitutions may have little effect on the properties of the protein, depending, for example, on the region of the protein in which the substitution is made.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising nucleic acids useful in the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.